ABSTRACTAlthough researchers have noted high level activation of rodent mononuclear phagocytes for nitric oxide (NO) synthase type 2 (S2) expression and NO production with a variety of agents such as interferon (IFN) gamma and endotoxin, it has been difficult to demonstrate activation of human mononuclear phagocytes. The purpose of this study was to determine if IFN-alpha serves as an activator in vitro and in vivo in humans. Treatment of normal monocytes or mononuclear cells in vitro with IFN-alpha caused a dose-dependent increase in monocyte NOS2 activity and NO production, and increased expression of NOS2 protein and mRNA expression. To determine if in vivo administration of IFN-alpha also modulated NOS2, we studied blood cells from patients with hepatitis C before and after IFN-alpha therapy. Untreated patients with chronic hepatitis C virus infection had levels of NOS activity and NOS2 antigen in freshly isolated mononuclear cells similar to those of healthy subjects, and they expressed minimal or no NOS2 mRNA. However, IFN-alpha treatment of patients with hepatitis C infection was associated with a significant elevation in mononuclear cell NOS activity, NOS2 antigen content, and NOS2 mRNA content. IFN-alpha-treated patients had significant decreases in levels of serum alanine aminotransferase and plasma hepatitis C mRNA. The degree of IFN-alpha-enhanced mononuclear cell NOS2 antigen content correlated significantly with the degree of reduction in serum alanine aminotransferase levels. Thus, IFN-alpha treatment of cells in vitro or administration of IFN-alpha to hepatitis C patients in vivo increases expression of mononuclear cell NOS2 mRNA expression, NOS activity, NOS2 antigen expression, and NO production. Since NO has been reported to have antiviral activity for a variety of viruses, we speculate that induced NO production may be related to the antiviral action(s) of IFN-alpha in hepatitis C infection.

Figure 3: (A–C) NOS enzyme activity in extracts of blood mononuclear cells (A), serum ALT (B), and plasma hepatitis C RNA levels (C). The bars show the mean + one SEM for samples taken from the different subject groups. Control, normal control subjects; Hep C, patients with hepatitis C not on therapy; Hep C + IFN, patients with hepatitis C on IFN-α therapy. For NOS measurements, n = 9 for Control, 18 for Hep C, and 15 for Hep C + IFN; for serum ALT, n = 15; and for plasma hepatitis C RNA, n = 4. (D) NOS enzyme activity in extracts of blood mononuclear cells from six individual patients before and after IFN-α treatment. The lines connect the values from an individual patient's samples before and after IFN-α therapy. The solid squares show the means ± one SEM of the groups.

Mentions:
In an attempt to determine if IFN-α treatment in vivo augmented NOS2 expression, we studied patients with hepatitis C before and after IFN-α therapy. Table 1 displays characteristics of the subjects and details of their treatments. As expected, hepatitis C patients receiving IFN-α treatment had higher levels of IFN-α than did normal subjects or hepatitis C patients not receiving IFN-α. There was an overall difference in the blood mononuclear cell NOS activity levels among the three groups (P <0.004; Fig. 3 A). Untreated patients with chronic hepatitis C and healthy controls had comparable NOS activity (ability to convert l-arginine to l-citrulline) in freshly isolated blood mononuclear cells. The activity was inhibited by >80% by inclusion of N-monomethyl-l-arginine in the assaying (data not shown), indicating that the activity was mediated by NOS. Patients receiving IFN-α2b therapy had significantly higher NOS activity levels than did healthy controls (adjusted P <0.05) and hepatitis C patients not receiving IFN-α2b therapy (adjusted P <0.05). Although IFN-α2b treatment caused increases in NOS2 levels, levels of serum alanine aminotransferase (ALT; an indicator of active hepatitis) and plasma hepatitis C RNA decreased with IFN-α2b therapy (Fig. 3, B and C; P = 0.002 and 0.02, respectively, by paired Students t test). When we analyzed samples from individuals both before and after receiving IFN-α2b, there was a significant increase in NOS activity after the IFN-α2b treatment in six patients in whom paired samples were available at baseline and during IFN-α2b therapy (2–8-wk time interval) (P <0.02, paired t test; Fig. 3 D).

Figure 3: (A–C) NOS enzyme activity in extracts of blood mononuclear cells (A), serum ALT (B), and plasma hepatitis C RNA levels (C). The bars show the mean + one SEM for samples taken from the different subject groups. Control, normal control subjects; Hep C, patients with hepatitis C not on therapy; Hep C + IFN, patients with hepatitis C on IFN-α therapy. For NOS measurements, n = 9 for Control, 18 for Hep C, and 15 for Hep C + IFN; for serum ALT, n = 15; and for plasma hepatitis C RNA, n = 4. (D) NOS enzyme activity in extracts of blood mononuclear cells from six individual patients before and after IFN-α treatment. The lines connect the values from an individual patient's samples before and after IFN-α therapy. The solid squares show the means ± one SEM of the groups.

Mentions:
In an attempt to determine if IFN-α treatment in vivo augmented NOS2 expression, we studied patients with hepatitis C before and after IFN-α therapy. Table 1 displays characteristics of the subjects and details of their treatments. As expected, hepatitis C patients receiving IFN-α treatment had higher levels of IFN-α than did normal subjects or hepatitis C patients not receiving IFN-α. There was an overall difference in the blood mononuclear cell NOS activity levels among the three groups (P <0.004; Fig. 3 A). Untreated patients with chronic hepatitis C and healthy controls had comparable NOS activity (ability to convert l-arginine to l-citrulline) in freshly isolated blood mononuclear cells. The activity was inhibited by >80% by inclusion of N-monomethyl-l-arginine in the assaying (data not shown), indicating that the activity was mediated by NOS. Patients receiving IFN-α2b therapy had significantly higher NOS activity levels than did healthy controls (adjusted P <0.05) and hepatitis C patients not receiving IFN-α2b therapy (adjusted P <0.05). Although IFN-α2b treatment caused increases in NOS2 levels, levels of serum alanine aminotransferase (ALT; an indicator of active hepatitis) and plasma hepatitis C RNA decreased with IFN-α2b therapy (Fig. 3, B and C; P = 0.002 and 0.02, respectively, by paired Students t test). When we analyzed samples from individuals both before and after receiving IFN-α2b, there was a significant increase in NOS activity after the IFN-α2b treatment in six patients in whom paired samples were available at baseline and during IFN-α2b therapy (2–8-wk time interval) (P <0.02, paired t test; Fig. 3 D).

Bottom Line:
Untreated patients with chronic hepatitis C virus infection had levels of NOS activity and NOS2 antigen in freshly isolated mononuclear cells similar to those of healthy subjects, and they expressed minimal or no NOS2 mRNA.IFN-alpha-treated patients had significant decreases in levels of serum alanine aminotransferase and plasma hepatitis C mRNA.The degree of IFN-alpha-enhanced mononuclear cell NOS2 antigen content correlated significantly with the degree of reduction in serum alanine aminotransferase levels.

ABSTRACTAlthough researchers have noted high level activation of rodent mononuclear phagocytes for nitric oxide (NO) synthase type 2 (S2) expression and NO production with a variety of agents such as interferon (IFN) gamma and endotoxin, it has been difficult to demonstrate activation of human mononuclear phagocytes. The purpose of this study was to determine if IFN-alpha serves as an activator in vitro and in vivo in humans. Treatment of normal monocytes or mononuclear cells in vitro with IFN-alpha caused a dose-dependent increase in monocyte NOS2 activity and NO production, and increased expression of NOS2 protein and mRNA expression. To determine if in vivo administration of IFN-alpha also modulated NOS2, we studied blood cells from patients with hepatitis C before and after IFN-alpha therapy. Untreated patients with chronic hepatitis C virus infection had levels of NOS activity and NOS2 antigen in freshly isolated mononuclear cells similar to those of healthy subjects, and they expressed minimal or no NOS2 mRNA. However, IFN-alpha treatment of patients with hepatitis C infection was associated with a significant elevation in mononuclear cell NOS activity, NOS2 antigen content, and NOS2 mRNA content. IFN-alpha-treated patients had significant decreases in levels of serum alanine aminotransferase and plasma hepatitis C mRNA. The degree of IFN-alpha-enhanced mononuclear cell NOS2 antigen content correlated significantly with the degree of reduction in serum alanine aminotransferase levels. Thus, IFN-alpha treatment of cells in vitro or administration of IFN-alpha to hepatitis C patients in vivo increases expression of mononuclear cell NOS2 mRNA expression, NOS activity, NOS2 antigen expression, and NO production. Since NO has been reported to have antiviral activity for a variety of viruses, we speculate that induced NO production may be related to the antiviral action(s) of IFN-alpha in hepatitis C infection.